Late stage counterscreen results for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based cell-based dose response assay for activators of the Pregnane X Receptor (PXR)

Transcription factors are critical regulators of gene expression (1). Under conditions such as environmental stress and exposure to endogenous toxins, transcription factors can rapidly modulate the transcription of genes whose products regulate cell proliferation and metabolism. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor of the basic helix-loop-helix protein superfamily involved in the biological response to aromatic hydrocarbons, and regulates the expression of xenobiotic-metabolizing enzymes such as cytochrome P450, aldehyde dehydrogenase, quinone reductase, and other phase I and phase II detoxification genes (2, 3). In response to various compounds, including the environmental pollutants dioxins, benzo(a)pyrene, dietary contaminants, grapefruit juice, endogenous toxins, and plant products such as carotinoids, nicotine and caffeine (2, 4-6), cytosolic AHR complexes with chaperones hsp90, p23, and XAP2, translocates to the nucleus where it dimerizes with the AHR nuclear translocator (ARNT) to influence target gene transcription (7, 8). Gain-of-function studies in mice reveal the oncogenic potential of AHR (9), while other reports show roles for AHR in diverse biologic events such as organ development (10, 11), immune function and allergy (12), and estrogen responsiveness (13). The identification of agonists of AHR will provide useful tools to elucidate the roles of this receptor in cell metabolism, transcriptional control, and tumor formation.

Assay Overview:The purpose of this assay is to identify compounds that act as agonists of the human ligand activated transcription factor, aryl hydrocarbon receptor (AHR). This cell-based assay measures the ability of compounds to activate AHR signaling. The assay employs human hepatoma (HepG2) cells stably transfected with the AHR-dependent pGudLuc6.1-DRE plasmid (HG2L6.1c3 cell line), which expresses the firefly luciferase reporter gene under control of a minimal promoter containing a synthetic dioxin response element (DRE) (14-16). Cells are incubated with test compounds for 24 hours, followed by cell lysis and detection of well luminescence using a commercially available luciferase reagent. As designed, compounds that act as AHR agonists will increase AHR activity and nuclear translocation, leading to increased activity of the DRE, increased transcription of the luciferase reporter gene, and increased well luminescence. Compounds are tested in singlicate at a final nominal concentration of 5.0 micromolar.Protocol Summary:The HG2L6.1c3 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Minimum Essential Medium (MEM) α supplemented with 10% v/v premium fetal bovine serum, 400 micrograms/mL G418, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).Prior to the start of the assay 5000 cells in a 4 microliter volume of assay media (growth media as above except without geneticin) were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 20 nL of test compound in DMSO (1.5 % final DMSO concentration), DMSO alone, or Indirubin (60 micromolar final concentration) to the appropriate wells. Next, the plates were incubated for 24 hours at 37 degrees C (5% CO2, 95% RH). After equilibrating the plates to room temperature for 30 minutes, the assay was stopped by dispensing 4microliters of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 5 minutes. Well luminescence was measured on the ViewLux plate reader.The percent activation for each compound was calculated using the following mathematical formula:% Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median__Low_Control ) )Where:Test_Compound is defined as wells containing test compound.Low_Control is defined as wells containing DMSO.High_Control is defined as wells containing Indirubin.A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.PubChem Activity Outcome and Score:The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.The PubChem Activity Score range for active compounds is 100-15, for inactive 15-0.List of Reagents:HG2L6.1c3 AHR cell line (provided by Assay provider)Indirubin (Amplachem, part Aa-31440)Minimum Essential Medium α (Invitrogen, part 12561072)G418 sulfate powder (Gemini Bio-Products, part 400-11P)Trypsin-EDTA solution (Invitrogen, part 15400054)Fetal Bovine Serum, Premium (Atlanta Biologicals, part S11150)100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)SteadyLite HTS Assay Kit (PerkinElmer, part# 6016989)T-175 tissue culture flasks (Corning, part 431080)Hyperflask cell culture flasks (Corning, part 10024)1536-well plates (Greiner, part 789072)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. In this assay, Indirubin had an EC50 of approximately 150nM. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.